An early warning system for dangerous breast cancer?

originally posted April 8, 2002

SAN FRANCISCO, CA - A tiny protein called
RhoC found in breast tumors may someday give doctors and patients
an early warning system that could spot dangerously aggressive
breast cancer long before it begins to spread, and identify
the need for aggressive treatment.

A test to detect the protein is still more than a year away
from clinical trials. But promising early results show that
RhoC(note: this link will open a second browser window)
can serve as a marker for breast tumors that are most likely
to spread, or metastasize - even identifying them when they're
less than a centimeter in diameter. (click
here to see images of breast tissue samples that have
been tested for RhoC. Note: this
link will open a second browser window).

Physicians from the University of Michigan Comprehensive
Cancer Center developed the test based on their prior research
on the RhoC gene, and proved its effectiveness in 182 tissue
samples from the U-M's breast cancer library. Results will
be presented April 9 at the meeting of the American
Association for Cancer Research.(Note: this link
will open a second browser window)

The test detected invasive cancer that had the potential
to metastasize with 88 percent specificity, and had 92 percent
specificity for tiny tumors that had already metastasized.
Samples of normal breast, benign breast cysts, or non-invasive
breast cancer had little RhoC.

"This is a very promising marker for small but invasive
breast cancers that may metastasize, which right now are hard
to identify," says lead author Celina Kleer, M.D., an
assistant professor of pathology at the U-M
Medical School(note: this link will open a second
browser window) who specializes in breast cancer.
"While more research is needed before clinical testing
can begin, we hope it will help identify early-stage cancer
that could be vulnerable to aggressive treatment, perhaps
with drugs that target Rho protein."

Kleer and her colleagues, including experienced RhoC researchers
Sofia Merajver, M.D., Ph.D., associate professor of internal
medicine, and Kenneth van Golen, Ph.D., assistant professor,
embarked on the study to find out how much RhoC was produced
in different kinds of breast cancer cells, compared with normal
breast cells.

Previously, (note:
this link will open a second browser window) they
had shown that the RhoC gene was overexpressed in inflammatory
breast cancer, a particularly deadly variety that grows and
metastasizes quickly. Overexpression of the gene, they believed,
might also occur in other kinds of aggressive breast cancer
- leading to larger quantities of the RhoC protein in cells
of those cancers.

RhoC, whose full name is RhoC-GTPase, is an enzyme involved
in changing the internal skeleton of a cell - changes that
allow a cell to polarize or move. That ability is important
in muscle cells, which produce a lot of RhoC. But in cancerous
non-muscle cells, RhoC is key to the structural changes that
give a cell the ability to break off from a tumor, float through
the body in the bloodstream, and take hold in a satellite
location - in other words, to metastasize.

In finding the inflammatory breast cancer correlation, the
U-M team was the first to show that RhoC, already implicated
in liver, pancreas and skin cancer, was also involved in breast
cancer. U-M researchers, led by Merajver, then showed that
transplanting the RhoC gene into normal breast cells in mice
transforms those cells into cancerous ones with metastatic
potential.

The new research started with the development of a RhoC test.
With help from the U-M Protein Structure Facility, and their
knowledge of the RhoC gene, the team created an antibody that
would latch on to RhoC protein anywhere in a tissue sample.
A stain specific to the antibody then allowed the U-M researchers
to see how concentrated the protein was in different tissue
samples from breast tumors and surrounding areas.

The breast cancer library at the Cancer Center provided 182
tissue samples from 164 patients whose breasts had been biopsied
at the U-M, as well as information about whether they had
cancer or benign breast disease like fibrocystic changes.
Samples of breast cancer tissue were accompanied by information
describing exactly what variety of cancer it was, how large
the tumor was, whether it was invasive (spreading beyond the
layer of cells where it started and into healthy tissue),
what stage it was in, and whether it had metastasized through
the body.

The cancerous samples included everything from ductal carcinoma
in situ (a low-grade localized condition), to stage IV invasive
ductal carcinomas that had spread to lymph nodes or other
parts of the body. The wide spectrum allowed the team to look
for differences between cancers of different sizes, stages,
types and levels of invasiveness.

Antibody staining revealed the expected high levels of RhoC
in muscle and blood vessel cells surrounding the breast tissue,
and none in normal breast tissue. But the concentrations of
the reddish-brown stain in the cancerous tissue varied greatly.
When Kleer correlated the samples that stained darkest with
their clinical characteristics, she discovered a connection.

"We found RhoC only in invasive cancers, and it almost
always correlated with the presence of metastases. Very few
non-metastatic cancers contained high levels of RhoC,"
she says. "The level of RhoC expression also increased
as the stage of the breast cancer increased, which is another
confirmation that it's a marker of more aggressive cancer.
We had enough samples from invasive metastatic cancers of
less than one centimeter in size to show that RhoC is highly
specific for those tumors, but we'd like to look at more samples
to be sure."

Kleer, Merajver, van Golen and their colleagues are preparing
to examine even more breast samples for the presence of RhoC,
to see if their initial results hold up. The team is also
in the process of planning clinical studies on the predictive
power of RhoC.

The study was funded by the National Institutes of Health,
the Department of Defense's breast cancer research program,
and a grant from the John and Suzanne Munn Endowment at the
U-M Comprehensive Cancer Center. In addition to Kleer, Merajver,
and van Golen, the study's authors are Zhi-Fen Wu, M.D.; Yanhong
Zhang, Ph.D.; and Mark Rubin, M.D.